Cytokines are small molecular weight proteins that have a myriad of biological functions (for background information, see Balkwill, F. R., et al., 1989, Immun. Today, 10:299). For example, cytokines are known to be capable of stimulating their own synthesis, as well as the production of other cytokines from a variety of cell types. They are also associated with disease. A good example is the presence of the cytokines interleukin-1 (IL-1) and tumor necrosis factor (TNF). IL-1 has been demonstrated to have multiple biological activities with the two prominent being fever production and lymphocyte activation. Moreover, both cytokines, alone or in combination, cause a shock state in animals that hemodynamically and hematologically is characteristic of septic shock in man caused by bacterial infection. TNF, in addition, has recently been shown to be involved in initiating the expression of human immunodeficiency virus in human cells that carry latent virus. Folks et al., 1989, PNAS (USA), 86:2365. TNF and IL-1 also play a role in various autoimmune diseases, particularly arthritis. Duff, et al. 1987, International Conference on Tumor Necrosis Factor and Related Cytotoxins, 175:10.
In addition to IL-1 and TNF, another cytokine, IL-6, has recently been shown to be involved in infection, particularly sepsis, as well as in affecting the growth of tumor cells. Hack, et al., 1989, Blood, 74:1704, and Miki et al, 1989, FEB, 250:607. IL-6 is also termed hybridoma growth factor, interferon-beta-2, B-cell stimulatory factor 2, 26 kD protein, and hepatocyte stimulating factor.
Adherence to an appropriate substratum has been shown to be important in transcriptional expression of cytokine mediators of inflammation produced by macrophages or monocytes, and adherence to different matrices has recently been shown to result in preferential gene induction (Sporn, S. A., et al., 1990, J. of Immun., 144:4434-4441; Thorens, B., et al., 1987, Cell, 48:671). For example, within 30 minutes of monocyte adherence to plastic, a complex set of regulatory events is initiated as defined by rapid changes of mRNA levels of several inflammatory mediators and proto-oncogenes (Haskill, S., et al., 1988, J. of Immunol., 140:1690). IL-1.beta.,TNF-.alpha. and c-fos are rapidly elevated, whereas CSF-1 steady state mRNA levels increase by 90 minutes. In contrast, expression of c-fms and lysozyme is rapidly down-regulated These genes are modulated by adherence to different biologically relevant substrates (Eierman, D. F., 1989, J. of Immunol., 14:1970-1970).
Although high steady state mRNA levels of important mediators of inflammation are rapidly induced by adherence, adherence by itself is insufficient to cause efficient translation and secretion of IL-1.beta.,TNF-.alpha., or CSF-1 (Haskill, S., et al., supra). Activation by a second signal, such as bacterial endotoxin, is required for the secretion of all three gene products. Thus, it is clear that signals derived from the act of adherence are likely to play a significant role in the activation and differentiation of monocytes allowing them to respond to infection and to influence the local tissue environment (Sporn, S. A, supra).
Recently, a protein termed NF-.kappa.B has been shown to be a transcriptional activator (Sen, R. and Baltimore, D., 1986, Cell, 4:705-716). This factor has been shown to bind to DNA regulatory regions of certain cytokine genes (Leonardo, M. and Baltimore, D., 1989, Cell, 58:227-229). Various agents cause the induction of nuclear NF-.kappa.B DNA-binding activity (Sen and Baltimore, supra). It is thus thought that NF-.kappa.B is a transcriptional regulator of gene expression for various cytokine genes. It would therefore be desirable to identify molecules that inhibit the effects of NF-.kappa.B since these would be useful to regulate the effects of cytokines in the inflammatory response.
It has recently been shown that NF-.kappa.B is associated with a 36 kD protein termed I.kappa.B (Baeurle, P. and Baltimore, D., 1988, Cell, 51:211-217; Baeurle, P. and Baltimore, D., 1988, Science, 242:540-546). NF-.kappa.B consists of proteins having molecular weights of 50 and 65 kD. I.kappa.B binds to the 65 kD subunit (Baeurle, P. and Baltimore, D., 1989, Genes and Development, 3:1689-1698). Finally, recent experimental evidence shows that phosphorylation of I.kappa.B blocks its inhibitory effect on DNA binding activity of NF-.kappa.B. This is consistent with the observation that protein kinases activate NF-.kappa.B DNA binding activity in vitro (Ghosh, S. and Baltimore, D., 1990, Nature, 344:678-682).
Because of the importance of I.kappa.B in regulating gene expression, it will be appreciated that the purification, cloning, and expression of this molecule will make available assays for identification of regulators of NF-.kappa.B and I.kappa.B that will have significant medical applications.